It is known that recombinant vectors, such as the feline herpes virus vector (FHV-1), may be used as a live carrier for developing vaccines against feline pathogens, e.g., feline leukemia virus, feline immunodeficiency virus, feline calicivirus, feline parvovirus, feline coronavirus and feline Chlamydia. See, for example, WO 94/0361, WO 91/01332 and Wardley, R. C., et al., J. of Gen. Virology (1992), 73, 1811-1818. However, the use of such recombinant FHV-1 vectors typically has been restricted to expressing feline leukemia (Wardley, R. C., et al., J. of Gen. Virology (1992), 73, 1811-1818) or feline infectious peritonitis disease virus antigens (PCT/EP94/02990 (WO 95/07987)). No successful attempts of using recombinant FHV-1 vectors for expressing antigenic determinants for the feline calicivirus disease has been known. While myriad influencing factors have been postulated, no one factor or set of factors has been identified as being the source of this failure.
Almost all gene sequences that encode proteins or polypeptides in eukaryotes are characterized as either coding (exon) or non-coding (intron) sequences. Introns are precisely spliced out of the initial gene transcript (pre-mRNA) before it is transported to the cytoplasm of the cell for translation. Sequences immediately bordering splice junctions are typically conserved in eukaryotic genes. Conserved junction sequences located between an exon and an intron are generally referred to as the 5' splice sites or donor sites. Sequences located at the boundary between an intron and an exon are generally referred to as the 3' splice sites, or acceptor sites. Further, short conserved sequences, referred to as branch point sequence, are typically located within the intron, usually 10 to 50 nucleotides upstream from an acceptor site. (See The RNA World, eds. R. F. Gesteland, J. F. Atkins, Cold Spring Harbor Laboratory Press (1993)).
So called consensus sequences typically represent basic sequences of nucleotides that are derived from a large set of observed similar sequences in a specific region of a nucleic acid molecule. (See Stenesh, J., Dictionary of Biochemistry and Molecular Biology, Second Ed., John Wiley & Sons (1989)). Known consensus sequences (DNA sequences) of the splicing signals include:
5' splice site or donor site: .sup.C.sub.A AG/GT.sup.A.sub.G AGT PA1 3' splice site or acceptor site: (.sup.T.sub.C).sub.9 NCAG/G, wherein N=A or G or T or C PA1 Branch point sequence: .sup.C.sub.T N.sup.C.sub.T T.sup.A.sub.G AC
(See, P. Senapathy, et al., Methods in Enzymology, Vol. 183, pp. 252-278 (1993)).
The calicivirus capsid (C) gene encodes the calicivirus capsid protein, which has been identified as an important antigen for developing vaccines for feline calicivirus disease. Although consensus DNA sequences, which are closely related to the splicing signals (donor, acceptor and branching sites) have been identified in genes coding for other eukaryotic proteins, as well as viruses that replicate in eukaryotes, no consensus DNA sequences for the calicivirus capsid (C) protein gene, including the calicivirus capsid (C) gene of FCV strain 2280, have been identified or isolated.
In nature, feline calicivirus genes are transcribed in the cytoplasm of feline calicivirus transformed or infected cells. (See, Hagan and Bruner's Microbiology and Infectious Diseases of Domestic Animals, eds. J F Timoney, J H Gillespie, F W Scott and J E Barlough, 8th edition, Comstock Publishing Ass. Cornell University Press (1988), (2nd Printing 1992)). As such, potential splicing signals, if any, are not accessible to the splicing machinery typically located in the nucleus of such infected cells. Thus, if any splicing signals exist, then they are not able to play a role in the processing of viral RNA.
On the other hand, transcription of FHV-1 genes occurs in the nucleus of infected cells and it has been observed that some of the transcribed RNAs are spliced. Further, foreign genes inserted in a recombinant FHV-1 virus (vector) are transcribed in the nucleus of the infected cells. As a consequence, the resulting RNAs are accessible to the splicing machinery of the infected cells.
Bovine respiratory syncytial virus (BRSV) is a viral pathogen whose genes are transcribed in the cytoplasm of infected cells. It has been reported that inactivation of splicing signals in the BRSV glycoprotein G gene, which normally could not be detected in bovine cells infected with a recombinant bovine herpesvirus 1 vector (BHV-1) containing the G gene, resulted in the expression of the gene in bovine cells infected by a recombinant BHV-1 virus having the mutated gene. See F. A Rijsewijk, R. C. Ruuls, K. Westerink and J. T. Van Oirschot, Department of Bovine Virology, Institute for Animal Science and Health, ID-DLO Lelystad, The Netherlands, at the 20.sup.th International Herpesvirus Workshop, Jul. 29-Aug. 3, 1995, University of Groningen, The Netherlands. However, the findings do not indicate, among other things, (1) the identity and position of the mutated splicing sites, (2) whether such splicing sites are present in the FCV C gene, or (3) whether the inactivation of such splicing sites, if present, would permit the complete or partial expression thereof in the nucleus of cells infected therewith.
Accordingly, there remains a need for recombinant DNA sequences, DNA molecules containing such DNA sequences that code for polypeptides or proteins that are naturally transcribed in the cytoplasm of cells and methods for making or using same. In particular, there remains a need for modified DNA sequences coding for the FCV capsid protein that are capable of being transcribed in the nucleus of eukaryotic cells without being altered by the cells' splicing machinery. Moreover, there remains a need for recombinant expression vectors that include such DNA sequences, cell cultures transformed or infected with such recombinant vectors and vaccines including such recombinant vectors and/or recombinant DNA sequences for the prevention and treatment of FCV disease.